RESEARCH ARTICLE Open Access

Complex sputum microbial composition in

Abstract Background: An increasing number of studies have implicated the microbiome in certain diseases, especially chronic diseases. In this study, the bacterial communities in the sputum of pulmonary tuberculosis patients were explored. Total DNA was extracted from sputum samples from 31 pulmonary tuberculosis patients and respiratory secretions of 24 healthy participants. The 16S rRNA V3 hyper-variable regions were amplified using bar-coded primers and pyro-sequenced using Roche 454 FLX. Results: The results showed that the microbiota in the sputum of pulmonary tuberculosis patients were more diverse than those of healthy participants (p<0.05). The sequences were classified into 24 phyla, all of which were found in pulmonary tuberculosis patients and 17 of which were found in healthy participants. Furthermore, many foreign bacteria, such as Stenotrophomonas, Cupriavidus, Pseudomonas, Thermus, Sphingomonas, Methylobacterium, Diaphorobacter, Comamonas, and Mobilicoccus, were unique to pulmonary tuberculosis patients. Conclusions: This study concluded that the microbial composition of the respiratory tract of pulmonary tuberculosis patients is more complicated than that of healthy participants, and many foreign bacteria were found in the sputum of pulmonary tuberculosis patients. The roles of these foreign bacteria in the onset or development of pulmonary tuberculosis shoud be considered by clinicians. Keywords: Pulmonary tuberculosis, Sputum, Microbiota, Diversity

Background across scales that range from molecular to cellular, to

Chronic pulmonary tuberculosis poses a global health whole organism and population levels [3].emergency. It has been known for many centuries and is The development of nucleotide sequencing has helpedmainly caused by the bacillus Mycobacterium tuberculosis. reveal the importance of microbiota to human health [4].Many reports have revealed co-infection with different For example, community and microbial ecology-basedstrains or species of Mycobacterium in pulmonary tuber- pathogenic theories have been introduced to explain theculosis patients. Mixed infection with Beijing and non- relationship between dental plaque and the host [5]. TheBeijing strains of M.tuberculosis [1] has been reported to urine microbiomes of men with sexually transmitted in-mediate the increased reinfection rate in regions with a fection were found to be dominated by fastidious, an-high incidence of tuberculosis. Similarly, MAC (Mycobac- aerobic and uncultivable bacteria [6]. Furthermore, theterium avium complex) and M.tuberculosis coexist in microbiota interact with nutrients and host biology tosome patients with combined mycobacterial infections [2]. modulate the risk of obesity and associated disorders,The systems biology concept of persistent infection is that including diabetes, obesity inflammation, liver diseasesinfectious diseases reflect an equilibrium between the host and bacterial vaginosis (BV) [7-10]. Patients with neo-and the pathogen that is established and maintained by a natal necrotising enterocolitis have lower microbiotabroad network of interactions. These interactions occur diversity, which is asscociated with an increase in the abundance of Gammaproteobacteria [11]. Ichinohe et al* Correspondence: tangsj1106@sina.com; xkguo@shsmu.edu.cn revealed that microbiota can regulate the immune defence2 Tuberculosis Centre for Diagnosis and Treatment, Shanghai Pulmonary against respiratory tract influenza A virus infection [12].Hospital, Tongji University School of Medicine, Shanghai 200433, China Ehlers and Kaufmann also emphasised the association1 Department of Medical Microbiology and Parasitology, Shanghai Jiao TongUniversity School of Medicine, 200025, Shanghai, China

2012 Cui et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Cui et al. BMC Microbiology 2012, 12:276 Page 2 of 8http://www.biomedcentral.com/1471-2180/12/276

between chronic diseases and dysbiosis or a disturbed Results and discussion

variability of the gut microbiome [13]. In light of the Resultsrecent discovery of cystic fibrosis associated lung Sequence diversitymicrobiota, Delhaes and Monchy et al discussed the The 454 pyro-sequencing method was used to analysis amicrobial community as a unique pathogenic entity total of 71928 PCR amplicons in the samples from pul-[14]. Huang and Lynch emphasised that microbiota, as monary tuberculosis patients and healthy participants,a collective entity, may contribute to pathophysiologic The amplicons averaged approximately 200 bp in length,processes associated with chronic airway disease [15]. and the average number of sequences per sample wasRobinson et al also suggested the conservation or res- 1307.8. The mean Shannon diversity and evenness indicestoration of the normal community structure and func- in the pulmonary tuberculosis samples were 6.1926 (SD,tion of host-associated microbiota should be included 0.8093) and 0.9615 (SD, 0.0177), respectively. Both indicesin the prevention and treatment of human disease [16]. were significantly higher than those in the healthy partici-In summary, microbiota are very important to human pants, which were 5.5145 (SD, 0.6545) (p=0.006) andhealth, Understanding the microbial composition in the 0.9341 (SD, 0.0216) (p=0.000) , respectively.respiratory tract of pulmonary tuberculosis patientsmay enhance our awareness of microbiota as a collect-ive entity or even collective pathogenic entity, and the Clustering analysis of the respiratory tract microbiota canrole this entity plays in the onset and development of separate healthy participants from pulmonary tuberculosispulmonary tuberculosis. patients In this work, we collected 31 sputum samples from The similarities between the respiratory tract secretionpulmonary tuberculosis patients from Shanghai Pulmon- microbiota of the healthy participants and sputum micro-ary Hospital, and 24 respiratory secretion samples biota of the pulmonary tuberculosis patients were esti-from healthy participants in Shanghai, China as con- mated by calculating UniFrac distances. Figure 1 showstrols, and investigated the composition of the micro- that the healthy participants were clustered together, whilebiota in the lower respiratory tract of pulmonary the pulmonary tuberculosis patients were divided intotuberculosis patients. several different sub-branches.

Figure 1 Bacterial communities grouped by individual. Each terminal branch represents the total bacterial community detected in one enrolled subject. All nodes were recovered at 100% using the Jackknife method. Names beginning with N represent samples from healthy participants, while those beginning with TB represent samples from patients with pulmonary tuberculosis.Cui et al. BMC Microbiology 2012, 12:276 Page 3 of 8http://www.biomedcentral.com/1471-2180/12/276

As shown in Figure 2, clustering after principal coordin- and Fervidicoccus were unique to and widespread amongate analysis (PCoA) of the UniFrac distance demonstrated the pulmonary tuberculosis patients.a strong clustering of healthy participants away from pul-monary tuberculosis patients. To better characterise the The phylum level composition of respiratory microbiomessputum microbiomes, the sequences were sorted to the A total of 24 phyla were detected in the pulmonary tuber-genera level. A total 614 genera were observed; 235 culosis samples, while 17 phyla were detected in healthygenera were observed in healthy participants, and 564 participants. Actinobacteria, Bacteroidetes, Proteobacteria,genera were found in pulmonary tuberculosis patients, and Crenarchaeota were widely and abundantly distrib-although more than half of these accounted for only a uted among nearly all of the samples. Firmicutes (37.02%),small fraction of the total sequences. As shown in Figure 3, Bacteroidetes (29.01%), Proteobacteria (16.37%), Crenarch-Streptococcus, Granulicatella, Actinomyces, Prevotella, and aeota (3.16%), and Actinobacteria (2.89%) were commonVeillonella were predominant in the microbiota of both in the healthy participants, while Firmicutes (41.62%), Bac-healthy participants and pulmonary tuberculosis patients. teroidetes (7.64%), Proteobacteria (17.99%), ActinobacteriaIn contrast, Anoxybacillus, Klebsiella, Acinetobacter, Pilibac- (21.20%), and Crenarchaeota (7.5%) were common in theter, Abiotrophia, Paucisalibacillus, and Rothia were more pulmonary tuberculosis patients. Chlamydiae, Chloroflexi,abundant in pulmonary tuberculosis patients than healthy Cyanobacteria/Chloroplast, Deinococcus-Thermus, Elusi-participants. Neisseria, Porphyromonas, TM7_genera_incer- microbia, Euryarchaeota, SR1, Spirochaetes, Synergistetes,tae_sedis, Parvimonas, Campylobacter, Haemophilus, and and Tenericutes were found in both the healthy partici-Fusobacterium were less common in pulmonary tubercu- pants and pulmonary tuberculosis patients, although theylosis patients than healthy participants. Furthermore, were rare in some samples. Aquificae, Caldiserica, Gem-Stenotrophomonas, Cupriavidus, Pseudomonas, Thermus, matimonadetes, Lentisphaerae, Planctomycetes, Thermo-Sphingomonas, Brevundimonas, Brevibacillus, Methylo- desulfobacteria, and Verrucomicrobia were unique to thebacterium, Diaphorobacter, Comamonas, Mobilicoccus, pulmonary tuberculosis samples. Moreover, in healthy

Figure 2 UniFrac community comparison of healthy participants and patients with pulmonary tuberculosis. The sputum microbiomes were clustered using un-weighted UniFrac. The percentage of variation explained by each principal component is indicated on the axis. The circles with names beginning with N represent samples from healthy participants, while those beginning with TB correspond to samples from patients with pulmonary tuberculosis.Cui et al. BMC Microbiology 2012, 12:276 Page 4 of 8http://www.biomedcentral.com/1471-2180/12/276

Figure 3 Hierarchical clustering of sputum microbial composition at the genus level. The names of some of the most abundant genera corresponding to terminal taxa depicted in the heatmap are listed to the right of the figure. Subjects listed at the top and right of the heatmap indicate microbiome and genus relationships, respectively. Names beginning with N represent samples from healthy participants, while those beginning with TB correspond to samples from patients with pulmonary tuberculosis.Cui et al. BMC Microbiology 2012, 12:276 Page 5 of 8http://www.biomedcentral.com/1471-2180/12/276

participants, Deinococcus-Thermus, Bacteroidetes, and Several rare genera were present in the sputum of pulmonaryFusobacteria accounted for 0.01%, 29.01% and 8.06%, re- tuberculosis patients, such as Thermus, Pelomonas, Methylo-spectively. However, in pulmonary tuberculosis patients, bacterium, Comamonas, Lactobacillus, Thermobacillus, Auri-Deinococcus-Thermus increased to 0.93%, Bacteroidetes, tidibacter, Lapillicoccus, and Devriesea.and Fusobacteria decreased to 7.64% and 1.35%, respectively. DiscussionSeveral genera were uniquel to the respiratory tracts of This study provides the first report on the microbialpulmonary tuberculosis patients composition of the lower respiratory tract of pulmonaryMany genera were unique to in the sputum of pulmonary tuberculosis patients through the amplification of 16Stuberculosis patients. As shown in Figure 3 and Table 1, rRNA V3 hyper-variable regions using bar-coded pri-Phenylobacterium, Stenotrophomonas, Cupriavidus, and mers and pyro-sequencing by Roche 454 FLX. ThePseudomonas were found in nearly half of the tuberculosis results revealed that the microbial composition of thepatients we enrolled; furthermore, their total copies lower respiratory tract in pulmonary tuberculosisaccounted for more than 1% of the total sequences from patients was more diverse (p<0.05) than in healthy parti-the sputum of pulmonary tuberculosis patients. Other cipants. Charlson et al reported that the microbial com-genera such as Sphingomonas, Mobilicoccus, Brevundimonas, position of saliva or pharynx secretions can reflect theBrevibacillus, and Diaphorobacter were much more widely microbial communities in the lower respiratory tract,detected in pulmonary tuberculosis patients, even though and their results showed that there is a topographicalthey accounted for only a small number of sequences. continuity of bacterial populations in the healthy human respiratory tract [17]. Therefore, we chose to use sputumTable 1 The distribution of some genera that were and respiratory secretions in this study. However, theuniquely found in the sputum of pulmonary tuberculosis best samples to use would be lung lavage fluid, whichpatients perfectly reflects the lower microbial composition of the Genera respiratory tract. However, obtaining lung lavage fluid isPhenylobacterium 13/31 2.15% challenging, especially from healthy volunteers, becauseStenotrophomonas 12/31 2.15% lung lavage is painful and may even be harmful. This may raise some ethical issues. In contrast, sputum and Cupriavidus 16/31 1.60% respiratory secretions are easily obtained through non- Caulobacter 5/31 1.56% invasive, patients-friendly collection methods. Therefore, Pseudomonas 15/31 1.27% we chose to analyse sputum and respiratory tract secre- Thermus 14/31 0.71% tions in our study. A previous study showed that fewer Sphingomonas 16/31 0.66% than 1% of commensal organisms are able to grow under Brevundimonas 17/31 0.49% laboratory conditions [18]; therefore, traditional cultivation- based strategies for analysing the complexity and genetic Pelomonas 15/31 0.47% diversity of microbial communities are strongly biased. Acidovorax 13/31 0.47% However, modern methods, based on barcoded primers Brevibacillus 16/31 0.36% and 454 pyro-sequencing allow for a thorough profilingMethylobacterium 13/31 0.34% of the microbiota of each enrolled person [19,20]. Pub- Diaphorobacter 17/31 0.31% lished studies have also proved that the 16S rRNA V3 Comamonas 14/31 0.26% region sequence ideally suited for distinguishing all bac- terial species to the genus level, except for closely Mobilicoccus 20/31 0.24% related Enterobacteriaceae [21]. Fervidicoccus 13/31 0.21% The lower respiratory tract microbiome of pulmonary Serpens 5/31 0.19% tuberculosis patients was distinct from that of the Lactobacillus 12/31 0.18% healthy participants. As shown in Figures 1 and 2, the Thermobacillus 12/31 0.16% pulmonary tuberculosis patients formed a clear cluster Auritidibacter 13/31 0.14% that was separate from the healthy participants based on their microbiota. The phyla Bacteroidetes and Fusobac- Deinococcus 9/31 0.13% tera were significantly underrpresented in pulmonary tu- Lapillicoccus 13/31 0.11% berculosis patients compared with healthy participants, Devriesea 13/31 0.11% while Actinobacteria was significantly overrepresented in: the number of pulmonary tuberculosis patients in whom sequences from pulmonary tuberculosis patients. Moreover, bacteriathe corresponding genera were found.: the percentage of sequences of the corresponding genera of all from the phylum Deinococcus-Thermus were widely dis-sequences found in pulmonary tuberculosis patients. tributed in pulmonary tuberculosis patients (15/31), butCui et al. BMC Microbiology 2012, 12:276 Page 6 of 8http://www.biomedcentral.com/1471-2180/12/276

rarely found in healthy participants, and the phyla Aqui- patients. This may be why the populations of many nor-ficae, Caldiserica, Gemmatimonadetes, Lentisphaerae, mal bacteria are decreased or absent from the micro-Planctomycetes, Thermodesulfobacteria and Verrucomi- biota of the pulmonary tuberculosis patients. At thecrobia were unique to pulmonary tuberculosis patients. same time, a strong host strong immune responseFigure 1 shows the genera Klebsiella, Pseudomonas and against the pathogen may damage or produce lesions inAcinetobacter were more common in pulmonary tuber- the lung tissue, and consequently the micro-culosis patients, and we postulated that these bacteria environment of the lower respiratory tract may favourmay aggravate the syndrome of pulmonary tuberculosis colonisation or even host invasion by foreign microor-in these patients. Table 1 shows that the genera Phenylo- ganisms. These foreign bacteria may cooperate with M.bacterium, Stenotrophomonas, Cupriavidus, Caulobacter, tuberculosis to cuase additional damage to the lung tis-Pseudomonas, Thermus and Sphingomonas were unique sue. In this model, although M. tuberculosis plays a cen-to and widely distributed in patients with pulmonary tral role in the disease, the other bacteria may assist intuberculosis. the destruction of the lung tissue, especially in active tu- The respiratory tract microbiota of pulmonary tuber- berculosis. If M. tuberculosis eliminated promptly, how-culosis patients, who suffer from chronic infection, ever, lung funtion can be restored. Further investigationmight be important in the pathogenicity of this disease. will be required to determine whether pulmonary tuber-The variety of bacterial genera especially the presence of culosis is the cause of increased foreign bacterial colon-some abnormal genera in the sputum of pulmonary tu- isation of the lower respiratory tract or vice versa (i.e.,berculosis patients suggested that the pulmonary tuber- the presence of foreign bacteria aggravates the symp-culosis patient lung is an ecological niche that can toms of pulmonary tuberculosis); is also possible thatsupport the growth of a high variety of bacteria, espe- both occur simultaneously.cially certain abnormal bacteria. These abnormal generareportedly widespread in the environment, and some of Conclusionsthem have even been reported to be associated with This study demonstrated that the microbial compositionsome infectious diseases [22-27]. Coenye et al also of the respiratory tract of pulmonary tuberculosisreported the isolation of unusual bacteria from the re- patients was more complicated than that of healthyspiratory secretions of cystic fibrosis patients [22]. How- volunteers, and many foreign bacteria were found in theever, there are few reports on whether these organisms sputum of pulmonary tuberculosis patients. These for-can cause human disease. The lower respiratory tract is eign bacteria may participate in the onset or develop-an open system and can communicate freely with the ment of pulmonary tuberculosis.environment. We speculated that, in pulmonary tuber-culosis patients, the lung micro-environment may be- Methodscome more susceptible to colonisation by some foreign All of the procedures for the collection and handling ofmicrobes. The host response to pathogens is charac- patient samples and data were reviewed and approvedterised by rapid recognition combined with strong innate by the ethics committee of the Shanghai Pulmonary(i.e., inflammatory) and adaptive immune responses, Hospital and Shanghai Jiaotong University School ofenabling microbial eradication often at the cost of sig- Medicine, incompliance with the Helsinki Declaration ofnificant tissue damage. Furthermore, the host is con- the World Medical Association. All study subjects pro-stantly facing the challenge of discriminating between vided written informed consent to participate in thesymbiotic and pathogenic bacteria to organise an appro- study.priately an adaptive response [28]. These responses leadto the extensive fibrosis associated with recurring infec- Specimenstions, possibly leading to a decreased clearance of lymph A total of 31 pulmonary tuberculosis patients, ranging inand lymph-associated particles from the infected region age 23 to 67 years old, with a age median of 39 years[29]. The lungs of individual patients typically contain and a male/female ratio of 19/12, were recruited fromdiverse lesions with varied overall structures that change the Shanghai Pneumonia Hospital. All patients were freeover time [3]. Ultimately, a strong host response to the of HIV. The patients were clinically diagnosed with pul-clearance of M. tuberculosis may produce local lesions in monary tuberculosis based on sputum smear, sputumthe lung. This may in turn increase the possibility that culture, and computed tomography results. The sputumforeign bacteria will colonise or grow in the lower re- samples were collected after the patients had beenspiratory tract. During the initial disease-causing inva- admitted to the hospital. A portion of the sputum sam-sion of the lung by M. tuberculosis, a strong host ple was used for medical tests, and the remaining spu-immune response may kill or clear some normal bacteria tum was preserved for DNA extraction after the patientsin the lower respiratory tract of pulmonary tuberculosis were confirmed to have pulmonary tuberculosis. Anti-Cui et al. BMC Microbiology 2012, 12:276 Page 7 of 8http://www.biomedcentral.com/1471-2180/12/276

TB drug treatment (isoniazid, rifampicin, ethambutol, sequence), while the sequence after the hyphen was usedpyrazinamide) and its efficiency as demonstrated by to amplify the sequences of the V3 end region.computed tomography, were also considered to confirm To ensure that a sufficient quantity of PCR productthe diagnosis of pulmonary tuberculosis. None of the was amplified, a two-step PCR strategy was used. Thepatients had taken antibiotics for at least 3 months be- first step was carried out in a 25 l reaction volume con-fore sampling. Of the 31 patients tested, 12 were sputum taining 2.5 l of PCR buffer (TAKARA), 0.625 U ExTaqculture positive, 9 were sputum smear positive, 20 were (TAKARA), 0.1 l of BSA (TAKARA), and 2 l of pri-clinically diagnosed with bilateral tuberculosis, 7 were mer solution with 100 mol of each forward and reverseclinically diagnosed with right pulmonary tuberculosis, 2 primer and 50 ng of extracted DNA as a template;were clinically diagnosed with left lung tuberculosis, 1 ddH2O was added to reach the final volume of the reac-was clinically diagnosed with tuberculosis pleurisy, and 1 tion. Touchdown PCR was performed as follows: 5 minwas clinically diagnosed with tuberculosis bronchiectasis. at 94C for initial denaturation, followed by 20 cycles of The healthy volunteers were recruited from the same 1 min at 94C for denaturation, 1 min at 65C forregion as the tuberculosis patients. A total of 24 healthy annealing and 1 minute at 72C for extension, with theparticipants, ranging from 38 to 66 years old, with a me- annealing temperature decreasing by 0.5C for eachdian age of 55, and a male and female ratio of 13/11, cycle. The reaction volume in the second step of thewere recruited from Shanghai, China. The volunteers PCR was 50 l and contained 5 l of the product fromhad similar lifestyle and eating habits, nutritional status step one as a template. The reaction also included 5 land physical condition, were free of basic pulmonary of PCR buffer (TAKARA), 1.25 U ExTaq (TAKARA), 0.2diseases, severe lung disease, severe oral disease, sys- l of BSA (TAKARA), 24 l of water and 200 mol oftemic disease and other known diseases such as obesity each barcoded forward and reverse primer. The amplifi-or diabetes, that could affect the microbial composition cation was carried out for five cycles of 1 minute at 94Cof the respiratory tract. Volunteers with a history of for denaturation, 1 minute at 55C for annealing, andsmoking or drinking were also excluded. The healthy 1 minute at 72C, with the temperature maintained atparticipants had not taken any antibiotics for at least 20C after the reaction was complete. Sequencing was3 months before sampling. The samples from healthy performed at the Chinese National Human Genomeparticipants were a mixture of saliva and pharyngeal Centre in Shanghai using a Roche 454 FLX instrument.secretions collected by deep coughing in the early morn- The resulting sequences were published as SRA acces-ing before gargling. By coughing, the community that sion SRA051957.was originally in the sputum was contaminated by thenormal flora of the oral cavity and pharynx. (The Phylogenetic and statistical analysisdetailed information of the pulmonary tuberculosis The datasets were taxonomically grouped using the RDPpatients and the healthy participants were showed in classifier (the naive Bayesian classifier of the RibosomalAdditional file 1). Database Project) at a confidence level of 90% [30]. The gross sequencing data were first searched for the linker, primers, and their reverse complements using the plat-Establishment of a pyro-sequencing library and pyro- form provided by the centre. The identified primersequencing using the 454 platform sequences were trimmed from each sequence read. Se-DNA extraction and PCR of the 16S rRNA V3 region quence reads that did not contain the 5-end primerwere performed as described in our previously published were removed from the dataset. The same program wasarticle [20]. However, several additional modifications also used for barcode identification. Barcodes were iden-were made. Fresh sputum samples were chosen soon tified within the first 25 bases of the reads. Sequenceafter routine tests confirmed the diagnosis of pulmonary reads were binned into FASTA files based on theirtuberculosis. After liquefaction at room temperature for barcodes.1 hour in a sterilised sodium hydroxide solution, 3 ml of Individual sequences were aligned using the Alignersample was aliquoted into three 1.5 ml Eppnedorf tubes, tool, and aligned sequences files for each sample werepasteurised at 83C for 30 min, and further extracted processed by complete linkage clustering using distanceusing a Bacterial DNA kit (OMEGA, Bio-Tek, USA). criteria. We used Uclust to cluster all of the sequences,PCR enrichment of the 16S rRNA V3 hyper-variable region with a cut-off value of 97%. After clustering, we used awas performed with the forward primer 5-XXXXXXXX- representative sequence of each type as the OUT (oper-TACGGGAGGCAGCAG-3 and the reverse primer 5-XX ational taxonomic units), and the record of each OUTXXXXXX-ATTACCGCGGCTGCTGG-3. The 5 terminus sequence included the number of sequences and theof each primer contained a different 8-base- oligonucleo- associated classification information. These data weretide tag (represented by XXXXXXXX in the primer used to calculate the Shannon diversity and evennessCui et al. BMC Microbiology 2012, 12:276 Page 8 of 8http://www.biomedcentral.com/1471-2180/12/276